Yutaka Ishida

Cloning and characterization of the D-tagatose 3-epimerase gene from Pseudomonas cichorii ST-24

Abstract The gene encoding d -tagatose 3-epimerase ( d -TE) from Pseudomonas cichorii ST-24 was cloned and sequenced. It was found to consist of 873 bp encoding 290 amino acid residues. The molecular weight of the deduced amino acid sequence of d -TE was determined to be 32.5 kDa. The deduced amino acid sequence showed no extensive homology with sequences of other sugar-related epimerases, but homology was observed with several hypothetical proteins of procaryotes, i.e. Synechocystis sp., Bacillus subtilis, Haemophilus influenzae , and Escherichia coli . The d -TE gene was expressed in E. coli .

Production of d-tagatose 3-epimerase of Pseudomonas cichorii ST-24 using recombinant Escherichia coli

Abstract The d -tagatose 3-epimerase ( d -TE) gene of Pseudomonas cichorii ST-24 was expressed in Escherichia coli under the control of the trc promoter. The d -TE production level was highest in E. coli JM105 as a host strain and in NZC medium as a culture medium. Production of d -TE by E. coli JM105 was about 100-fold higher than that of d -TE by P. cichorii ST-24, and the enzyme constituted ∼5% of the total protein. d -TE from E. coli JM105 was purified by polyethylene glycol precipitation, DEAE-Toyopearl 650M column chromatography, and Sephadex G-150 column chromatography. The overall purification procedure resulted in 16.7-fold purification with 18.2% recovery. The molecular weight of the purified d -TE was estimated to be 33.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and agreed with that of the d -TE from P. cichorii ST-24. d -TE produced by E. coli JM105 had generally the same enzymological properties, i.e. , the optimum pH and temperature, pH and thermal stabilities, and K m value, as that from Pseudomonas sp. ST-24, but the V max value of d -TE from E. coli was 6 times higher than that from Pseudomonas sp. ST-24. The N-terminal amino acid sequence of the recombinant d -TE agreed with that of the d -TE from P. cichorii ST-24.

Direct Production of Allitol from D-Fructose by a Coupling Reaction Using D-Tagatose 3-Epimerase, Ribitol Dehydrogenase and Formate Dehydrogenase.

Allitol was produced from D-fructose via a new NADH-regenerating enzymatic reaction system using D-tagatose 3-epimerase (D-TE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH). D-fructose was epimerized to D-psicose by the D-TE of Pseudomonas cichorii ST-24 and the D-psicose was subsequently reduced to allitol by the RDH of an RDH-constitutive mutant, X-22, derived from Klebsiella pneumoniae IFO 3321. NADH regeneration for the reduction of D-psicose by the RDH was achieved by the irreversible formate dehydrogenase reaction, which allowed the D-psicose produced from d-fructose to be successively transformed to allitol with a production yield from D-fructose of almost 100%. The reactions progressed without any by-product formation. After separation of the product from the reaction mixture by a simple procedure, a crystal of allitol was obtained in a yield exceeding 90%. This crystal was characterized and determined to be allitol by HPLC analysis, its IR and NMR spectra, its melting point, and optical rotation measurement.

d-Psicose induces upregulation of defense-related genes and resistance in rice against bacterial blight

We examined rice responses to a rare sugar, d-psicose. Rice growth was inhibited by d-psicose but not by common sugars. Microarray analysis revealed that d-psicose treatment caused an upregulation of many defense-related genes in rice, and dose-dependent upregulation of these genes was confirmed by quantitative reverse-transcription polymerase chain reaction. The level of upregulation of defense-related genes by d-psicose was low compared with that by d-allose, which is another rare sugar known to confer induction of resistance to rice bacterial blight in rice. Treatment with d-psicose conferred resistance to bacterial blight in rice in a dose-dependent manner, and the results indicate that d-psicose might be a candidate plant activator for reducing disease development in rice.

A rare sugar, D-allose, confers resistance to rice bacterial blight with upregulation of defense-related genes in Oryza sativa.

We investigated responses of rice plant to three rare sugars, d-altrose, d-sorbose, and d-allose, due to establishment of mass production methods for these rare sugars. Root growth and shoot growth were significantly inhibited by d-allose but not by the other rare sugars. A large-scale gene expression analysis using a rice microarray revealed that d-allose treatment causes a high upregulation of many defense-related, pathogenesis-related (PR) protein genes in rice. The PR protein genes were not upregulated by other rare sugars. Furthermore, d-allose treatment of rice plants conferred limited resistance of the rice against the pathogen Xanthomonas oryzae pv. oryzae but the other tested sugars did not. These results indicate that d-allose has a growth inhibitory effect but might prove to be a candidate elicitor for reducing disease development in rice.

Phosphorylation of D-allose by hexokinase involved in regulation of OsABF1 expression for growth inhibition in Oryza sativa L.

We previously reported that a rare sugar d-allose, which is the d-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). d-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that d-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, d-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The d-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, d-mannoheptulose (MNH). Consistently, d-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. d-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1S177A, indicating that the d-allose phosphorylation by HXK to d-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as d-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to d-allose in rice. These findings indicated that the phosphorylation of d-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for d-allose-induced growth inhibition.

The rare sugar d-allose acts as a triggering molecule of rice defence via ROS generation

Only d-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to d-allose. d-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-d-allose, a structural derivative of d-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding d-allose kinase to increase d-allose 6-phosphate synthesis were more sensitive to d-allose, but E. coli AlsI encoding d-allose 6-phosphate isomerase expression to decrease d-allose 6-phosphate reduced sensitivity. A d-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, d-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of d-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of d-allose to d-allose 6-phosphate, and treatment with d-allose might prove to be useful for reducing disease development in rice.

Rare sugar d-allose suppresses gibberellin signaling through hexokinase-dependent pathway in Oryza sativa L.

One of the rare sugars, d-allose, which is the epimer of d-glucose at C3, has an inhibitory effect on rice growth, but the molecular mechanisms of the growth inhibition by d-allose were unknown. The growth inhibition caused by d-allose was prevented by treatment with hexokinase inhibitors, d-mannoheptulose and N-acetyl-d-glucosamine. Furthermore, the Arabidopsisglucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1, showed a d-allose-insensitive phenotype. d-Allose strongly inhibited the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half rice seeds. The growth of the slenderrice1 (slr1) mutant, which exhibits a constitutive gibberellin-responsive phenotype, was also inhibited by d-allose, and the growth inhibition of the slr1 mutant by d-allose was also prevented by d-mannoheptulose treatment. The expressions of gibberellin-responsive genes were down-regulated by d-allose treatment, and the down-regulations of gibberellin-responsive genes were also prevented by d-mannoheptulose treatment. These findings reveal that d-allose inhibits the gibberellin-signaling through a hexokinase-dependent pathway.

Cloning and characterization of a polygalacturonase-encoding gene from Penicillium janthinellum

Abstract The genomic DNA encoding a polygalacturonase (PG) from the filamentous fungus Penicillium janthinellum was cloned by genome walking using single specific primer-polymerase chain reaction (SSP-PCR), and the complete nucleotide sequence was identified. The PG gene consists of 1266-bp encoding a protein of 371 amino acids, interrupted by two introns of 88 and 62-bp in length. Comparison of the amino acid sequences revealed the presence of a high degree of homology among PGs from different fungi. When the P. janthinellum PG gene was introducted into Aspergillus oryzae, the transformants showed about three-fold enhanced PG activity relative to the recipient parent strain, indicating that the gene encoded in the cloned DNA fragment was the fuctional PG gene.

A possibility of rare sugar applications for agro-usages

単糖とは一般的にブドウ糖(D-glucose)と果糖(D-fructose) を指すことが多く,これらを中心としたいくつかの糖で,自 然界の単糖の大部分が占められている.これに対して,その 存在量が極めて少ない単糖を「希少糖」と呼ぶ.希少糖の定 義は,国際希少糖学会によって「自然界にその存在量が少な い単糖とその誘導体」とされ,単糖と糖アルコールを合わせ ると,60種弱がこれまでに知られている.さらに,これら の各種誘導体を加えていくとその数は無数になるが,希少糖 の生産手法は限られていたため,希少糖のほとんどはこれま で長く入手できない状況にあった. しかしながら,1994年に香川大学農学部の何森らの研究 グループが,D-fructoseをD-allulose(=D-psicose)に変換 する酵素を生産する微生物の単離に成功し,希少糖生産へ の扉が大きく開かれた .何森らは,この酵素(D-tagatose 3-epimerase, DTE)を用いて,希少糖であるD-alluloseの大 量生産技術の確立を進め,さらに各種希少糖を体系的に生産 するために,単糖の構造と酵素反応・有機反応をベースとし た生産の設計図を模式化した「イズモリング(Izumoring)」 (図1)を発表した .これにより,何森を中心とした香川 DOI: 10.1584/jpestics.W17-35